The present invention relates to oligonucleotide probes and methods for the detection of target nucleic acid sequences in a sample. More particularly, the present invention relates to oligonucleotide probes which are internally cleavable when hybridized to target nucleic acid sequences, such cleavage leading to both signal generation and amplification by recycling.
The identification of a target nucleic acid sequence is of great importance in both biological research and medical diagnostics. Detection of a target sequence can be used to identify and/or type a specific DNA or RNA molecule and to uncover mutations.
Numerous methods and techniques exist in the art with which detection and/or identification of a target sequence can be effected. For example, polynucleotide sequencing methods can be used to determine the nucleotide sequence of a target DNA or RNA molecule. The methods typically used for sequencing include the Sanger dideoxy method, see, e.g., Sanger et al. (1977) Proc. Natl. Acad. Sci. USA, 74:5463-5467, or the Maxam and Gilbert method, see, e.g., Maxam et al, (1980) Methods in Enzymology, 65:499-559.
The polymerase chain reaction (PCR) can also be used to detect the presence of a target sequence in a sample. PCR utilizes oligonucleotide primers which specifically bind regions within the target sequence to amplify the target nucleic acid sequence, the generation of amplification products is indicative of the presence of the target sequence.
Another approach to target nucleic acid identification involves hybridizing an oligonucleotide probe to the target nucleic acid sequence wherein hybridization is indicative of the presence thereof.
An oligonucleotide probe binds to a target nucleic acid by forming hydrogen bonds between bases in the target and the oligonucleotide. Common B-DNA has conventional adenine-thymine (A-T) and guanine-cytosine (G-C) Watson and Crick base pairs with two and three hydrogen bonds formed therebetween, respectively. Conventional hybridization technology is based upon the capability of sequence-specific DNA or RNA oligonucleotide probes to bind to a complementary target nucleic acid via Watson-Crick hydrogen bonds. However, other types of internucleotide hydrogen bonding patterns are known wherein atoms not involved in Watson-Crick base pairing to a first nucleotide can form hydrogen bonds to another nucleotide. For example, thymine (T) can bind to an AT Watson-Crick base pair via hydrogen bonds to the adenine, thereby forming a T-AT base triad. Hoogsteen (1959, Acta Crystallographica 12:822) first described the alternate hydrogen bonds present in T-AT and C-GC base triads. More recently, G-TA base triads, wherein guanine can hydrogen bond with a central thymine, have been observed (Griffin et al., 1989, Science 245:967-971).
Oligonucleotide probes which can bind to a target nucleic acid with both Watson-Crick and non-Watson-Crick hydrogen bonds form extremely stable complexes with the target nucleic acid and as such have a variety of research and diagnostic utilities.
For example, oligonucleotides can be used as probes for target nucleic acids that are immobilized onto a filter or membrane, or are present in tissues, e.g. as described in Sambrook et al., (1989, Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY). However, the utility of linear oligonucleotide probes is frequently limited by their poor binding stability and selectivity.
Another example includes solution phase detection methods. Several solution-phase detection methods, sometimes referred to as homogeneous assays, are known. The term “homogeneous” is used in the art to refer to methods performed without separating unhybridized oligonucleotide probes from probe-target hybrids. These methods often rely upon the fact that the fluorescence of many fluorescent labels can be affected by the conformation of the oligonucleotide probe or by the immediate chemical environment.
U.S. Pat. No. 5,876,930 to Livak et al. discloses a method for identifying a target nucleic acid sequence. The method utilizes an oligonucleotide probe which includes a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of the reporter molecule. The oligonucleotide probe according to this method is constructed such that the probe exists in at least one single-stranded conformation when unhybridized, where the quencher molecule is near enough to the reporter molecule to quench the fluorescence of the reporter molecule. The oligonucleotide probe also exists in at least one conformation when hybridized to a target nucleic acid where the quencher molecule is not positioned close enough to the reporter molecule to quench the fluorescence of the reporter molecule. By adopting these hybridized and unhybridized conformations, the reporter molecule and quencher molecule on the probe exhibit different fluorescence signal intensities when the probe is hybridized and unhybridized. As a result, this method enables to determine the presence of a specific target nucleic acid sequence based on a change in the fluorescence intensity of the reporter molecule, the quencher molecule, or a combination thereof. The limitation of this approach is that no signal amplification is enabled, resulting in inability of detecting low target concentrations. In addition, this method is inherently characterized by a high background signal.
U.S. Pat. No. 5,925,517 to Tyagi et al. discloses unimolecular and bimolecular hybridization probes for the detection of nucleic acid target sequences. The probes include a target complement sequence, an affinity pair holding the probe in a closed conformation in the absence of target sequence, and either a label pair that interacts when the probe is in the closed conformation or, for certain unimolecular probes, a non-interactive label. Hybridization of the target and target complement sequences shifts the probe to an open conformation. The shift is detectable due to reduced interaction of the label pair or by decreasing a signal from a non-inverted label. Certain unimolecular probes can discriminate between target and non-target sequences differing by as little as one nucleotide. The limitation of this approach is that no signal amplification is enabled, resulting in inability of detecting low target concentrations. In addition, this method is inherently characterized by a high background signal.
U.S. Pat. No. 5,866,336 to Nazarenko et al. describes labeled nucleic acid amplification oligonucleotides, which can be linear or hairpin primers or blocking oligonucleotides. The oligonucleotides disclosed by Nazarenko are labeled with donor and/or acceptor moieties of molecular energy transfer pairs. The moieties can be fluorophores, such that fluorescent energy emitted by the donor is absorbed by the acceptor. The acceptor may be a fluorophore that fluoresces at a wavelength different from the donor moiety, or it may be a quencher. These oligonucleotides are configured so that a donor moiety and an acceptor moiety are incorporated into the amplification product. The invention also provides methods and kits for directly detecting amplification products employing the nucleic acid amplification primers. When labeled linear primers are used, treatment with exonuclease or by using specific temperature eliminates the need for separation of unincorporated primers. This “closed-tube” format greatly reduces the possibility of carryover contamination with amplification products, provides for high throughput of samples, and may be totally automated.
U.S. Pat. No. 4,766,062 to Diamond et al. describes a diagnostic reagent containing a complex of a probe polynucleotide bound via purine/pyrimidine hydrogen bonding to a labeled polynucleotide. The probe contains a target binding region capable of binding to a target sequence of a biological sample. U.S. Pat. No. 4,766,062 further describes a method in which contact with a sample containing the target nucleotide sequence causes binding, initially between the target and a single-stranded portion of the target binding region. Thereafter the labeled polynucleotide is displaced from the complex of branch migration of into the binding region. Determination of displaced labeled polynucleotide gives a value which is a function of the presence and concentration of target nucleotide sequence in the sample.
U.S. Pat. No. 5,451,503 to Hogan et al., which is discussed in more detail in the preferred embodiments section that follows, teaches of nucleic acid hybridization probes having at least one nucleic acid strand which has at least two separate target specific regions that hybridize to a target nucleic acid sequence, and at least two distinct arm regions that do not hybridize with the target nucleic acid but possess complementary regions that are capable of hybridizing with one another. These regions are designed such that, under appropriate hybridization conditions, the complementary arm regions will not hybridize to one another in the absence of the target nucleic acid; but, in the presence of target nucleic acid the target-specific regions of the probe will anneal to the target nucleic acid, and the complementary arm regions will anneal to one another, thereby forming a branched nucleic acid structure which is useful for target nucleic acid sequence detection.
Although the above mentioned methods are less complicated to perform than simple oligonucleotide probe detection methods such as that described by Sambrook et al. in which oligonucleotide probes are used to target nucleic acids that are immobilized onto a filter or membrane, some limitations still apply. For example, a method which is simple to perform such as that described by Livak et al. can yield false positive results since hybridization to non-target sequences will also yield, in some cases, a positive result.
In general, the above methods are characterized by low signal and high background. Hogan et al. teaches signal amplification by template recycling and background reduction by appropriate selection of the length of the arm regions of the oligonucleotides employed thereby. Methods which are aimed at producing more accurate results are oftentimes more complicated to perform. However, as is further shown and exemplified hereinunder, improved methods for signal amplification an background reduction are still required.
Thus, the present invention discloses novel oligonucleotides utilizable in a homogeneous detection method of target nucleic acid sequences, which method depends on the generation of a specific cleavage site in a hybridized, conformationaly changed oligonucleotide, and as such the present invention provides a simple method of target nucleic acid detection while retaining a high level of specificity.